Chromosomal replication of Escherichia coli is regulated and coordinated to the bacterial cell cycle. The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. Among the numerous proteins that are involved in the process of DNA replication, DnaA protein of 466 amino acid residues is unique in its specific role in the initiation process. As a sequence- specific DNA binding protein, it binds to sites in the chromosomal origin, oriC, the site where chromosomal replication begins. On binding, it then mediates a series of events that establishes the initiation of DNA replication. By a novel genetic method, a large collection of mutants has been isolated and characterized. We hope to identify residues essential for activity of DnaA protein in DNA replication. We will continue genetic and biochemical characterization of these novel dnaA mutants. Three of four aims are proposed under this general goal. The first specific aim is to identify the DNA binding domain of DnaA protein, and residues involved in this activity. The second aim is to determine the functional significance of a P-loop motif, a sequence highly conserved in homologs of dnaA and also found in many nucleotide binding proteins. Biochemical characterization of isolated P-loop mutants, investigation of the dominant-negative effect these mutants have when in a host that contains the dnaA+ allele, mutagenesis of the P-loop, and crosslinking studies are proposed. In addition, the collection of mutants may contain those that are defective in essential functions other than DNA binding and ATP binding. As a third aim, a biochemical study of these mutants is proposed. These studies will hopefully provide more insight into the function of DnaA protein in the initiation process. As a fourth aim, we plan to identify proteins that interact physically with DnaA protein by its use as an affinity ligand. Findings from these studies address the issue of how the replication apparatus assembles at the E. coli chromosomal origin, and whether other proteins interact physically to modulate the activity of DnaA protein. Included in this aim is a study to determine the stoichiometry of DnaA, DnaB, and DnaC proteins in isolated prepriming complexes. Collectively, these studies promise further insight on the role of DnaA protein in the initiation process, and provide a model system whereby this process may occur in other organisms.